Modern methods for the detection, enumeration and identification of microorganisms fall into two main categories. The first provides analysis after preliminary growth on a special nutrient media. The second category does not require preliminary growth. Methods utilized in the first category utilize several different chemical, biochemical, physical or optical techniques and require many hours or days for preliminary growth in order to produce enough homogeneous cells or colonies for detection. The second category utilizes methods of microscopy, flow cytometry or polymerase chain reaction (PCR). These methods allow for analysis immediately after sampling and sample treatment. Analyzing a single cell without preliminary growth belongs to these rapid micromethods.
Microscopy (light and fluorescent, visual or automated) and flow cytometry (absorbent, fluorescent or scattering) requires treatment of cells by absorbent or fluorescent dyes. Utilization of antibodies with attached fluorescent molecules helps in rapid identification of single cells. A higher concentration of colored molecules increases the reliability of analysis.
Well known markers for detection and identification of cells are artificial substrates, i.e., non-colored or fluorescent substances cleaved by enzymes or enzymatic groups with the production of light-absorbent or fluorescent molecules. Artificial substrates are broadly used for the detection of live microorganisms, detection by unique enzymes, identification by enzymatic profiles, or utilization in enzyme immunological analysis (EIA). Some artificial substrates produce non-soluble intracellular precipitates (e.g. tetrazolium salts, fluorescein-based substrates in acidic environments, Rezorufin and others). This feature is useful for microscopy and flow cytometry because of the production of specifically colored cell bodies. Other substrates produce soluble derivatives that are excreted from the cells and color the buffer solution (e.g. 4-Methylumbelliferone, tetramethylbenzidine, fluorescein in alkaline environments and others). This group of artificial substrates is able to produce a large amount of absorbent or fluorescent molecules because they do not accumulate in cells and do not interfere with biochemical pathways of living cells the way precipitates do.
Retention of fluorescent or absorbent molecules excreted from cells or produced in EIA in a small space around a single cell can easily create detectable concentrations of these molecules. Utilization of this useful feature of soluble absorbent or fluorescent molecules together with a simple hand-held device for cell sampling and then immediately treating cells for detection or identification purposes is the subject of the present invention.
Simple and rapid detection, enumeration and identification of single prokaryotic or eukaryotic cells is very important in medical microbiology, cytology, environmental science, detection of pollutant microorganisms in food, in the pharmaceutical industry, epidemiology, public and military defense, scientific research and other areas.